Anna Woloszyk

Nanodentistry: combining nanostructured materials and stem cells for dental tissue regeneration.

Regenerative dentistry represents an attractive multidisciplinary therapeutic approach that complements traditional restorative/surgery techniques and benefits from recent advances in stem cell biology, molecular biology, genomics and proteomics. Materials science is important in such advances to move regenerative dentistry from the laboratory to the clinic. The design of novel nanostructured materials, such as biomimetic matrices and scaffolds for controlling cell fate and differentiation, and nanoparticles for diagnostics, imaging and targeted treatment, is needed. The combination of nanotechnology, which allows the creation of sophisticated materials with exquisite fine structural detail, and stem cell biology turns out to be increasingly useful in regenerative medicine. The administration to patients of dynamic biological agents comprising stem cells, bioactive scaffolds and/or nanoparticles will certainly increase the regenerative impact of dental pathological tissues. This overview briefly describes some of the actual benefits and future possibilities of nanomaterials in the emerging field of stem cell-based regenerative dentistry.

Influence of the mechanical environment on the engineering of mineralised tissues using human dental pulp stem cells and silk fibroin scaffolds

Teeth constitute a promising source of stem cells that can be used for tissue engineering and regenerative medicine purposes. Bone loss in the craniofacial complex due to pathological conditions and severe injuries could be treated with new materials combined with human dental pulp stem cells (hDPSCs) that have the same embryonic origin as craniofacial bones. Optimising combinations of scaffolds, cells, growth factors and culture conditions still remains a great challenge. In the present study, we evaluate the mineralisation potential of hDPSCs seeded on porous silk fibroin scaffolds in a mechanically dynamic environment provided by spinner flask bioreactors. Cell-seeded scaffolds were cultured in either standard or osteogenic media in both static and dynamic conditions for 47 days. Histological analysis and micro-computed tomography of the samples showed low levels of mineralisation when samples were cultured in static conditions (0.16±0.1 BV/TV%), while their culture in a dynamic environment with osteogenic medium and weekly µCT scans (4.9±1.6 BV/TV%) significantly increased the formation of homogeneously mineralised structures, which was also confirmed by the elevated calcium levels (4.5±1.0 vs. 8.8±1.7 mg/mL). Molecular analysis of the samples showed that the expression of tooth correlated genes such as Dentin Sialophosphoprotein and Nestin were downregulated by a factor of 6.7 and 7.4, respectively, in hDPSCs when cultured in presence of osteogenic medium. This finding indicates that hDPSCs are able to adopt a non-dental identity by changing the culture conditions only. Also an increased expression of Osteocalcin (1.4x) and Collagen type I (1.7x) was found after culture under mechanically dynamic conditions in control medium. In conclusion, the combination of hDPSCs and silk scaffolds cultured under mechanical loading in spinner flask bioreactors could offer a novel and promising approach for bone tissue engineering where appropriate and rapid bone regeneration in mechanically loaded tissues is required.

Human Dental Pulp Stem Cells and Gingival Fibroblasts Seeded into Silk Fibroin Scaffolds Have the Same Ability in Attracting Vessels

Neovascularization is one of the most important processes during tissue repair and regeneration. Current healing approaches based on the use of biomaterials combined with stem cells in critical-size bone defects fail due to the insufficient implant vascularization and integration into the host tissues. Therefore, here we studied the attraction, ingrowth, and distribution of blood vessels from the chicken embryo chorioallantoic membrane into implanted silk fibroin scaffolds seeded with either human dental pulp stem cells or human gingival fibroblasts. Perfusion capacity was evaluated by non-invasive in vivo Magnetic Resonance Imaging while the number and density of blood vessels were measured by histomorphometry. Our results demonstrate that human dental pulp stem cells and gingival fibroblasts possess equal abilities in attracting vessels within silk fibroin scaffolds. Additionally, the prolonged in vitro pre-incubation period of these two cell populations favors the homogeneous distribution of vessels within silk fibroin scaffolds, which further improves implant survival and guarantees successful healing and regeneration.

The effect of extracellular acidosis on the behaviour of mesenchymal stem cells in vitro.

The stem cell fraction of a cell population is finely tuned by stimuli from the external microenvironment. Among these stimuli, a decrease of extracellular pH (pHe) may occur in a variety of physiological and pathological conditions, including hypoxia and inflammation. In this study, by using bone marrow stem cells and dental pulp stem cells, we provided evidence that extracellular acidosis endows the maintenance of stemness in mesenchymal cells. Indeed, continuous exposure for 21 d to low pHe (6.5-6.8) conditions impaired the osteogenic differentiation of both cell types. Moreover, the exposure to low pHe, for 1 and up to 7 d, induced the expression of stemness-related genes and proteins, drove cells to reside in the quiescent G0 alert state and enhanced their ability to form floating spheres. The pre-conditioning with extracellular acidosis for 7 d did not affect the differentiation potential of dental pulp stem cells since, when the cells were cultured again at physiological pHe, their multilineage potential was almost unmodified. Our data provided evidence of the role of extracellular acidosis as a modulator of the stemness of mesenchymal cells. This condition is commonly found both in systemic and local bone conditions, hence underlining the relevance of this phenomenon for a better comprehension of bone healing and regeneration.

Three-Dimensional imaging of the developing vasculature within stem cell-seeded scaffolds cultured in ovo

Successful tissue engineering requires functional vascularization of the three-dimensional constructs with the aim to serve as implants for tissue replacement and regeneration. The survival of the implant is only possible if the supply of oxygen and nutrients by developing capillaries from the host is established. The chorioallantoic membrane (CAM) assay is a valuable tool to study the ingrowth and distribution of vessels into scaffolds composed by appropriate biomaterials and stem cell populations that are used in cell-based regenerative approaches. The developing vasculature of chicken embryos within cell-seeded scaffolds can be visualized with microcomputed tomography after intravenous injection of MicroFil®, which is a radiopaque contrast agent. Here, we provide a step-by-step protocol for the seeding of stem cells into silk fibroin scaffolds, the CAM culture conditions, the procedure of MicroFil® perfusion, and finally the microcomputed tomography scanning. Three-dimensional imaging of the vascularized tissue engineered constructs provides an important analytical tool for studying the potential of cell seeded scaffolds to attract vessels and form vascular networks, as well as for analyzing the number, density, length, branching, and diameter of vessels. This in ovo method can greatly help to screen implants that will be used for tissue regeneration purposes before their in vivo testing, thereby reducing the amount of animals needed for pre-clinical studies.

Odyssey of human dental pulp stem cells and their remarkable ability to survive in extremely adverse conditions

Adult tissues contain stem cells, which proliferate to compensate for tissue loss throughout the life of the organism (Li and Clevers, 2010; Jimenez-Rojo et al., 2012). Therefore, adult stem cells are important for tissue repair and regeneration after injury. In seriously injured or carious teeth, stem cells residing in the dental pulp are responsible for the repair and regeneration of the damaged dental tissues (Caton et al., 2011; Mitsiadis et al., 2011). Dental pulp stem cells (DPSCs) can be isolated after labeling of the pulp cells with fluorescent mesenchymal stem cell (MSC) markers such as STRO-1, CD146, and CD44, followed by fluorescence-activated cell sorting (FACS) (Mitsiadis et al., 2012). Additional identification procedures include morphological criteria, adherence properties, proliferation, and differentiation potential, as well as tissue repair abilities of the sorted stem cell populations. The potential of DPSCs for the regeneration of bone tissue in patients has been demonstrated with a successful trial assay that has been realized in Napoli (Italy) several years ago (Caton et al., 2011; Mitsiadis et al., 2012). We have established a close scientific collaboration with the Napoli stem cell team headed by Prof. Gianpaolo Papaccio for the exchange of knowledge and materials. Several months ago, we have requested for a bunch of freshly isolated DPSCs in order to complete a number of experiments that we are currently realizing in our laboratories in Zurich (Switzerland). DPSCs were shipped with an express delivery company on the 26/11/2014 from Napoli with destination to Zurich. The shipped DPSCs were grown as a monolayer in a 25 cm2 culture flask containing the standard MSC culture medium. The flask that was sealed with Parafilm and left in ambient temperature should normally arrive to the destination the next day, but for unknown reasons the journey of DPSCs was longer and more exciting than expected. The DPSCs have followed an astonishing itinerary from Italy, to France (Paris), Great Britain, South Africa (Johannesburg), back to France, thereafter to Spain, France again. From France the cells finally arrived to Zurich Switzerland through Basel on the 4/12/2014. This itinerary can be considered as an Odyssey of DPSCs, which had to develop sophisticated mechanisms for their survival under these extremely adverse conditions. Sometimes discoveries are arising by chance, following a mistake or having an unexpected behavior. It was expected that the flask, upon its late arrival, would contain only dead cells. However, surprisingly, DPSCs (or a big part of them) were still alive after their journey (Figure ​(Figure1A).1A). DPSCs were extremely proliferative and became confluent within 3 days, after changing the medium (Figures 1B,C). How this could be explained? Recent findings indicate that quiescent (reserve) and proliferating (active) stem cell pools may coexist in separate but adjacent compartments of many tissues (Li and Clevers, 2010). It has been proposed that stem cells can adopt a reversible quiescence state characterized by reduced metabolic activity in conditions such as lack of oxygen and/or nutrients (Cheung and Rando, 2013). However, the mechanisms that allow quiescent stem cells to survive metabolic or environmental stress, to preserve their cellular and genomic integrity and to assure long-term survival are not yet elucidated. It has been suggested that quiescence is an actively maintained state regulated by intrinsic mechanisms to sustain metabolic function during persistent environmental stress, and thus ensuring stem cell survival (Cheung and Rando, 2013). Viable stem cells were found in post-mortem tissues (Latil et al., 2012). These cells were able to maintain their functional properties after prolonged storage in anoxia in vitro and after transplantation (Latil et al., 2012). Similarly, it has been shown that several stem cell populations reside in poorly oxygenated niches (Simsek et al., 2010). Quiescent stem cells have the ability to sense environmental changes and respond by re-entering the cell cycle for proliferation (Cheung and Rando, 2013). Severe hypoxia could be critical for maintaining the viability of DPSCs. It is possible that mechanisms compatible with the low metabolic state of quiescence have allowed rapid responses for DMSCs re-activation. Figure 1 Visualization of human dental pulp stem cells (hDPSCs): (A) upon arrival to Zurich (first passage), (B) after 6 h of culture (second passage), and (C) after 3 days of culture (second passage). Scale bars indicate the magnifications. This Odyssey revealed that DPSCs are able to survive for prolonged periods of time in conditions of extreme stress.

Cellular self-assembly into 3D microtissues enhances the angiogenic activity and functional neovascularization capacity of human cardiopoietic stem cells

While cell therapy has been proposed as next-generation therapy to treat the diseased heart, current strategies display only limited clinical efficacy. Besides the ongoing quest for the ideal cell type, in particular the very low retention rate of single-cell (SC) suspensions after delivery remains a major problem. To improve cellular retention, cellular self-assembly into 3D microtissues (MTs) prior to transplantation has emerged as an encouraging alternative. Importantly, 3D-MTs have also been reported to enhance the angiogenic activity and neovascularization potential of stem cells. Therefore, here using the chorioallantoic membrane (CAM) assay we comprehensively evaluate the impact of cell format (SCs versus 3D-MTs) on the angiogenic potential of human cardiopoietic stem cells, a promising second-generation cell type for cardiac repair. Biodegradable collagen scaffolds were seeded with human cardiopoietic stem cells, either as SCs or as 3D-MTs generated by using a modified hanging drop method. Thereafter, seeded scaffolds were placed on the CAM of living chicken embryos and analyzed for their perfusion capacity in vivo using magnetic resonance imaging assessment which was then linked to a longitudinal histomorphometric ex vivo analysis comprising blood vessel density and characteristics such as shape and size. Cellular self-assembly into 3D-MTs led to a significant increase of vessel density mainly driven by a higher number of neo-capillary formation. In contrast, SC-seeded scaffolds displayed a higher frequency of larger neo-vessels resulting in an overall 1.76-fold higher total vessel area (TVA). Importantly, despite that larger TVA in SC-seeded group, the mean perfusion capacity (MPC) was comparable between groups, therefore suggesting functional superiority together with an enhanced perfusion efficacy of the neo-vessels in 3D-MT-seeded scaffolds. This was further underlined by a 1.64-fold higher perfusion ratio when relating MPC to TVA. Our study shows that cellular self-assembly of human cardiopoietic stem cells into 3D-MTs substantially enhances their overall angiogenic potential and their functional neovascularization capacity. Hence, the concept of 3D-MTs may be considered to increase the therapeutic efficacy of future cell therapy concepts.

Iodixanol as a contrast agent in a fibrin hydrogel for endodontic applications

The application of biomaterials used in regenerative endodontics should be traceable. In this study, we checked some basic effects of rendering a fibrin hydrogel radiopaque using an iodine-based contrast agent (iodixanol) approved for systemic application. Fibrin hydrogels were prepared from a fibrin sealant (Tisseel) using either an isotonic iodixanol solution (Visipaque 320, test) or Tris buffer (control) as a diluent. Gelation kinetics, radiopacity, and swelling of lyophilized hydrogels were tested using standard methods. Hydrogel structure was evaluated using scanning electron microscopy (SEM). Furthermore, iodixanol release from the test gels was assessed using spectrophotometry, and tissue compatibility was compared between test and control hydrogels using the chick chorioallantoic membrane (CAM) assay. Results were compared using pairwise t-test, p < 0.05. Iodixanol caused a 70-fold delay in gelation to 26 min in the test compared to the control hydrogels (22 ± 1 s). Radiopacity of the test gels was 1.9 ± 0.2 mm Al/mm, compared to zero in the control hydrogels. Lyophilized hydrogel swelling was strongly reduced when iodixanol was added to the hydrogel (p < 0.05). Test hydrogels had an altered SEM appearance compared to controls, and exhibited a reduced porosity. Iodixanol release from the test hydrogels reached 14.5 ± 0.5% after 120 h and then ceased. This release did not have any apparent toxic effect and neither affected the viability, nor the physiology or vascularization of the CAM of fertilized chicken eggs. Iodixanol can render a fibrin hydrogel radiopaque and maintains its tissue compatibility, yet impacts gelation kinetics and hydrogel porosity.

Angiogenesis within Stem Cell–Seeded Silk Scaffolds Cultured on the Chorioallantoic Membrane and Visualized by 3D Imaging

The long-term survival and successful integration of implants for tissue replacement and regeneration highly depends upon the fast ingrowth of blood vessels from the surrounding tissues. Before selecting potential biomaterials for clinical applications, they must be thoroughly tested with proper analytical tools. This unit provides a protocol for studying the potential of cell-seeded scaffolds to attract vessels that will form vascular networks within biomaterials. It includes seeding of stem cells into silk fibroin scaffolds, angiogenesis assay on the chorioallantoic membrane (CAM) of fertilized chicken eggs, a procedure for perfusion with MicroFil, and finally microcomputed tomography (µCT) scanning. This technique can help screen potential biomaterial implants, thereby reducing the amount of animals needed for pre-clinical in vivo studies.